Antimicrobial and radioprotective compounds

ABSTRACT

The present invention relates to a method of treatment and/or prophylaxis of a microbial infection, comprising the step of administering an effective amount of a compound of formula (I), in which X and Y are either the same or different and selected from a heteroatom; is a double or single bond depending on the heteroatoms X and Y; R 1  to R 5  are either the same or different and selected from hydrogen or a non-deleterious substituent; and R 6  and R 7  are either the same or different and selected from hydrogen and a non-deleterious substituent or one of R 6  and R 7  are absent when there is a double bond present, pharmaceutically acceptable salts or derivatives, pro-drugs, tautomers and/or isomers thereof. The present invention also relates to a method for protecting a subject from radiation damage, a method of cancer radiotherapy and use as an antimicrobial or radioprotective agent of the compound of formula (I) defined above. Some of the compounds of formula (I) are novel and are also described in the present invention, together with pharmaceutical or veterinary compositions containing them.

The present application is a continuation of U.S. patent applicationSer. No. 11/923,404 filed Oct. 24, 2007, which was a continuation ofU.S. patent application Ser. No. 10/481,667 filed Aug. 26, 2004, whichwas a nationalization of Intl. Patent Appl. No. PCT/AU02/00783 filedJun. 14, 2002, that claimed priority to Russian Provisional Patent Appl.No. 2001/117033, filed Jun. 18, 2001, the entire contents of each ofwhich is specifically incorporated herein by reference in its entiretyby express reference thereto.

This invention relates to compounds which have antimicrobial andradioprotective activity. In particular, the invention relates tosubstituted nitrostyrene compounds which have activity against a widespectrum of organisms including bacteria, fungi and protozoa. Thecompounds of the invention also have the ability to provide protectionfrom radiation damage.

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art, in Australia or in any othercountry.

Bacterial, fungal and protozoal pathogens are responsible for a verywide variety of infections, ranging from minor respiratory ailments tofulminant systemic infections and chronic illnesses. Food poisoningcaused by organisms such as Salmonella or Campylobacter is common, andis often associated with endemic infection in livestock or poultryraised using intensive animal husbandry techniques.

Despite the wide availability of antibiotics, control of infection isdifficult, and many organisms have the ability to develop resistance.Many microorganisms cause problems which have hitherto proved to bequite intractable, such as multi-drug resistant Staphylococcus aureusinfection in hospitals, drug-resistant Enterococcus infections,bacterial, fungal and protozoal infection in HIV patients, tuberculosis,and malaria and other endemic infections in underdeveloped countries.

Currently there are only very few agents which have a wide spectrum ofactivity against pathogens of bacterial, fungal and protozoal origin.Antibiotics are the most widely used agents in the fight againstpathogenic microorganisms. However, most antibiotics have narrowspecificity. Even broad spectrum antibacterial antibiotics are not veryeffective against fungi and protozoa. Most antibiotics belong to arestricted range of classes of compounds; although improvedsemi-synthetic derivatives of these have developed, only a few newantibiotic compound classes have become available in the last twentyyears.

The choice of agents for protection of living organisms againstradioactive radiation is also quite limited. Among the radiationprotectors the most effective are sulphur-containing compounds (Kuna,1989). For example, cystamine is approved for use as aradiation-protective agent (Vladimirov et al, 1989). The index ofprotection of this preparation does not exceed 1.45, and has thedisadvantage that it causes diarrhoea. Another knownradiation-protective preparation is mercamine (β-mercaptoethylamine)(Mashkovskiy, 1986).

This has a low therapeutic index, short period of action (0.5-1 h), andshort duration of radiation protecting activity (15-30 min).

It is known that β-nitrostyrene and some of its derivatives demonstratebiological, and partly fungicidal activity (Foyer, 1973). Russian PatentNo. 2145215 showed that certain derivatives of arylnitroalkenes haveantimicrobial, antifungal, antiprotozoal activity, and are able toprovide protection from radiation damage. These compounds have thefollowing formula

in which R₁′ is H or CH₃; and

R₂′ and R₃′ are the same or different and are selected from H, OCH₃, OH,NO₂ and (CH₃)₂N.

The activities of these compounds are satisfactory, but there is a needfor low cost, low-toxicity agents with a wide spectrum of antimicrobialactivities.

We have now found that certain substituted nitrostyrene compounds haveexcellent activity against very wide spectrum of organisms, includingbacteria, fungi and protozoa and also have the ability to provideprotection from radiation damage.

SUMMARY OF THE INVENTION

The invention provides a method of treatment and/or prophylaxis of amicrobial infection, comprising the step of administering an effectiveamount of a compound of formula I:

in which

X and Y are either the same or different and selected from a heteroatom;

is a double or single bond depending on the heteroatoms X and Y;

R₁ to R₅ are either the same or different and selected from hydrogen ora non-deleterious substituent; and

R₆ and R₇ are either the same or different and selected from hydrogenand a non-deleterious substituent or one of R₆ and R₇ are absent whenthere is a double bond present,

pharmaceutically acceptable salts or derivatives, pro-drugs, tautomersand/or isomers thereof.

The invention also provides use of the compound of formula I in themanufacture of a medicament for the treatment and/or prophylaxis of amicrobial infection.

The invention further provides use of the compound of formula I for thetreatment and/or prophylaxis of a microbial infection.

The invention still further provides a method for protecting a subjectfrom radiation damage which comprises administering an effective amountof the compound of formula I to a subject in need thereof.

In another aspect, the invention provides a method of cancerradiotherapy which comprises administering to a subject in need of suchtherapy an effective amount of the compound of formula I and subjectingthe locus of a tumour in the subject to a radiation source.

In a further aspect, the invention provides use of the compound offormula I as an antimicrobial or radioprotective agent.

Preferably X and Y are either the same or different and selected from Oand N, more preferably both X and Y are oxygen.

Preferably R₁ and R₂ are either the same or different and selected fromhydrogen, hydroxy, halogen or optionally substituted C₁₋₆ alkyl.

R₃ to R₅ are preferably either the same or different and selected fromhydrogen, hydroxy, halogen, nitro, C₁₋₆ alkoxy or optionally substitutedC₁₋₆ alkyl.

Preferably halogen is chlorine or bromine.

The E isomer of the compounds of formula I is preferred.

Particularly preferred are compounds of the formula I in which X, Y,

, R₆ and R₇ are as defined above; R₁ and R₂ are either the same ordifferent and selected from hydrogen, hydroxy, Cl, Br and C₁₋₄ alkyl;and R₃ to R₅ are either the same or different and selected fromhydrogen, hydroxy, Cl, Br, nitro, C₁₋₄ alkoxy or C₁₋₄ alkyl.

Specific examples of the compounds of the present invention are asfollows:

-   -   (1) X and Y are O, R₁ is methyl and R₂ and R₃ are hydrogen        (3,4-methylenedioxy-β-methyl-β-nitrostyrene)

-   -   (2) X and Y are O and R₁ to R₃ are hydrogen        (3,4-methylenedioxy-β-nitrostyrene)

-   -   (3) X is N, Y is NH, R₁ is methyl and R₂ and R₃ are hydrogen        (benzimidazole-5-β-nitropropylene)

-   -   (4) X is N, Y is NH, R₁ is hydrogen, R₂ is methyl and R₃ is        absent (2-methyl benzimidazole-5-β-nitroethylene)

-   -   (5) X is O, Y is N, R₁ and R₂ are hydrogen and R₃ is absent        (benzoxazole-5-β-nitroethylene)

-   -   (6) X is N, Y is O, R₁ and R₂ are methyl and R₃ is absent        (2-methyl benzoxazole-5-β-nitropropylene)

Some of the compounds of the formula I are novel per se.

Accordingly, the invention provides a compound of formula Ia:

in which X, Y,

and R₁ to R₇ are as defined in formula I above,

with the provisos that when both X and Y are O and R₂ to R₇ arehydrogen, then R₁ is not hydrogen, C₁₋₄ alkyl or CO₂Et or when both Xand Y are O, then R₁ to R₇ are not hydrogen.

The invention also provides a process for the preparation of thecompound of formula Ia defined above which comprises condensing acompound of formula II:

in which X, Y,

, R₃ to R₇ are as defined in formula Ia above

with a compound of formula III:R₁R₂CHNO₂  III

in which R₁ and R₂ are as defined in formula Ia above.

The invention further provides a process for the preparation of thecompound of formula Ia defined above which comprises reacting a compoundof formula IV:

in which X, Y,

, R₁ to R₇ are as defined in formula Ia above with C(NO₃)₄.

The processes are preferably performed in the presence of a catalyst,such as, an amine or an alkali metal hydroxide, for example, NaOH orKOH.

In a further aspect, the invention provides a pharmaceutical orveterinary composition comprising the compound of formula Ia definedabove together with a pharmaceutically or veterinarily acceptablecarrier.

Preferably, the pharmaceutical or veterinary composition is a topical,oral or parenteral composition.

The pharmaceutically or veterinarily acceptable carrier is preferably anorganic solvent such as acetone, benzene, acetonitrile, DMSO or analcohol, for example, methanol or ethanol. While the compounds of thepresent invention show a poor solubility in water, when water iscombined with an organic solvent a stable mixture is formed.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

The term “heteroatom” denotes O, N or S.

The term “non-deleterious substituent” is used herein in its broadestsense and refers to a substituent which does not have a deleteriouseffect on the antimicrobial or radioprotective properties of thecompound. Examples include alkyl, alkenyl, alkynyl, aryl, halo,haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy,alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy,nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl,nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino,alkynylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl,alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy,alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, heterocycloxy,heterocyclamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl,carboalkoxy, carboaryloxy mercapto, alkylthio, arylthio, acylthio andphosphorus-containing compounds.

Particularly suitable non-deleterious substituents are alkyl, alkenyl,alkynyl, halo, haloalkyl, haloalkenyl, haloalkynyl, hydroxy, alkoxy,alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, nitroalkyl, nitroalkenyland nitroalkynyl.

In a preferred embodiment the non-deleterious substituents are C₁₋₆alkyl, halo, hydroxy, C₁₋₆ alkoxy and nitro.

The term “optionally substituted” means that a group may or may not befurther substituted with, for example, the groups specified above underthe definition of non-deleterious substituent.

The term “halogen” refers to fluorine, chlorine, bromine and iodine,preferably chlorine and bromine.

The term “alkoxy” is used herein in its broadest sense and refers tostraight chain, branched chain or cyclic oxy-containing radicals eachhaving alkyl portions, preferably C₁₋₆ alkyl, more preferably C₁₋₄alkyl. Examples of such alkoxy groups are methoxy, ethoxy, propoxy,butoxy and t-butoxy.

The terms “C₁₋₄ alkyl” or “C₁₋₆ alkyl” used either alone or in compoundwords such as “optionally substituted C₁₋₄ or C₁₋₆ alkyl” refer tostraight chain, branched chain or cyclic hydrocarbon groups having from1 to 6 carbon atoms. Illustrative of such alkyl groups are methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, neopentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl.

The salts of the compound of formula I or Ia are preferablypharmaceutically acceptable, but it will be appreciated thatnon-pharmaceutically acceptable salts also fall within the scope of thepresent invention, since these are useful as intermediates in thepreparation of pharmaceutically acceptable salts. Examples ofpharmaceutically acceptable salts include salts of pharmaceuticallyacceptable cations such as sodium, potassium, lithium, calcium,magnesium, ammonium and alkylammonium; acid addition salts ofpharmaceutically acceptable inorganic acids such as hydrochloric,orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric,sulfamic and hydrobromic acids; or salts of pharmaceutically acceptableorganic acids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulphonic,trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicylic,sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic,lauric, pantothenic, tannic, ascorbic and valeric acids.

In addition, some of the compounds of the present invention may formsolvates with water or common organic solvents. Such solvates areencompassed within the scope of the invention.

By “pharmaceutically acceptable derivative” is meant anypharmaceutically acceptable salt, hydrate or any other compound which,upon administration to the subject, is capable of providing (directly orindirectly) a compound of formula I or Ia or an antimicrobial orradioprotective active metabolite or residue thereof.

The term “pro-drug” is used herein in its broadest sense to includethose compounds which are converted in vivo to compounds of formula I orIa.

The term “tautomer” is used herein in its broadest sense to includecompounds of formula I or Ia which are capable of existing in a state ofequilibrium between two isomeric forms. Such compounds may differ in thebond connecting two atoms or groups and the position of these atoms orgroups in the compound.

The term “isomer” is used herein in its broadest sense and includesstructural, geometric and stereo isomers. As the compound of formula Ior Ia may have one or more chiral centres, it is capable of existing inenantiomeric forms.

The term “microbial infection” is used herein in its broadest sense andrefers to any infection caused by a microorganism and includes bacterialinfections, fungal infections, yeast infections and protozoalinfections.

The term “microorganism” includes any microscopic organism ortaxonomically related macroscopic organism within the categories algae,bacteria, fungi, yeast and protozoa or the like.

Bacterial infections include, but are not limited to, infections causedby Bacillus cereus, Bacillus anthracis, Clostridium botulinum,Clostridium difficile, Clostridium tetani, Clostridium perfringens,Corynebacteria diphtheriae, Enterococcus (Streptococcus D), Listeriamonocytogenes, Pneumoccoccal infections (Streptococcus pneumoniae),Staphylococcal infections and Streptococcal infections; Gram Negativebacteria including Bacteroides, Bordetella pertussis, Brucella,Campylobacter infections, enterohaemorrhagic Escherichia coli (EHEC/E.coli 0157:H7) enteroinvasive Escherichia coli (EIEC), enterotoxigenicEscherichia coli (ETEC), Haemophilus influenzae, Helicobacter pylori,Klebsiella pneumoniae, Legionella spp., Moraxella catarrhalis, Neisseriagonnorrhoeae, Neisseria meningitidis, Proteus spp., Pseudomonasaeruginosa, Salmonella spp., Shigella spp., Vibrio cholera and Yersinia;acid fast bacteria including Mycobacterium tuberculosis, Mycobacteriumavium-intracellulare, Myobacterium johnei, Mycobacterium leprae,atypical bacteria, Chlamydia, Mycoplasma, Rickettsia, Spirochetes,Treponema pallidum, Borrelia recurrentis, Borrelia burgdorfii andLeptospira icterohemorrhagiae and other miscellaneous bacteria,including Actinomyces and Nocardia.

Fungal infections include, but are not limited to, infections caused byAlternaria alternata, Aspergillus flavus, Aspergillus fumigatus,Aspergillus nidulans, Aspergillus niger, Aspergillus versicolor,Blastomyces dermatiditis, Candida albicans, Candida dubliensis, Candidakrusei, Candida parapsilosis, Candida tropicalis, Candida glabrata,Coccidioides immitis, Cryptococcus neoformans, Epidermophyton floccosum,Histoplasma capsulatum, Malassezia furfur, Microsporum canis, Mucorspp., Paracoccidioides brasiliensis, Penicillium marneffei, Pityrosporumovale, Pneumocystis carinii, Sporothrix schenkii, Trichophyton rubrum,Trichophyton interdigitale, Trichosporon beigelii and Rhodotorula spp.

Yeast infections include, but are not limited to, infections caused byBrettanomyces clausenii, Brettanomyces custerii, Brettanomycesanomalous, Brettanomyces naardenensis, Candida himilis, Candidaintermedia, Candida saki, Candida solani, Candida tropicalis, Candidaversatilis, Candida bechii, Candida famata, Candida lipolytica, Candidastellata, Candida vini, Debaromyces hansenii, Dekkera intermedia,Dekkera bruxellensis, Geotrichium sandidum, Hansenula fabiani,Hanseniaspora uvarum, Hansenula anomala, Hanseniaspora guillermondiiHanseniaspora vinae, Kluyveromyces lactis, Kloekera apiculata,Kluveromyces marxianus, Kluyveromyces fragilis, Metschikowiapulcherrima, Pichia guilliermodii, Pichia orientalis, Pichia fermentans,Pichia memranefaciens, Rhodotorula Saccharomyces bayanus, Saccharomycescerevisiae, Saccharomyces dairiensis Saccharomyces exigus, Saccharomycesuinsporus, Saccharomyces uvarum, Saccharomyces oleaginosus,Saccharomyces boulardii, Saccharomycodies ludwigii, Schizosaccharomycespombe, Torulaspora delbruekii, Torulopsis stellata, Zygoaccharomycesbailli and Zygosaccharomyces rouxii.

Protozoal infections include, but are not limited to, infections causedby Leishmania, Toxoplasma, Plasmodia, Theileria, Anaplasma, Giardia,Trichomonas, Trypanosoma, Coccidia, and Babesia. Specific examplesinclude Trypanosoma cruzi, Eimeria tenella, Plasmodium falciparum,Plasmodium vivax or Plasmodium ovale.

Preferably, the microbial infection is an infection caused by either aGram Positive or a Gram negative bacterium, for example, Staphylococcusaureus, Enterococcus fecalis, Klebsiella pneumonia, Salmonellatyphimurium or pseudotuberculosis, Acinetobacter, Pseudomonasaeruginosa, Clostridium perfringens, Clostridium difficile,Campylobacter jejuni or Bacteroides fragilis; a fungal or yeastinfection, for example, Trichophyton interdigitale; Aspergillusfumigatus or Candida albicans; or a protozoal infection, for examplePlasmodium falciparum or Trichomonas vaginalis.

Examples of microbial infections include bacterial or fungal woundinfections, mucosal infections, enteric infections, septic conditions,pneumonia, trachoma, ornithosis, trichomoniasis, fungal infections andsalmonellosis, especially in veterinary practice. The compounds of theinvention may also be used for the treatment of resistant microbialspecies or in various fields where antiseptic treatment or disinfectionof materials is required, for example, surface disinfection.

The term “subject” as used herein refers to any animal having a diseaseor condition which requires treatment with a pharmaceutically-activeagent. The subject may be a mammal, preferably a human, or may be adomestic or companion animal. While it is particularly contemplated thatthe compounds of the invention are suitable for use in medical treatmentof humans, it is also applicable to veterinary treatment, includingtreatment of companion animals such as dogs and cats, and domesticanimals such as horses, ponies, donkeys, mules, llama, alpaca, pigs,cattle and sheep, or zoo animals such as primates, felids, canids,bovids, and ungulates.

Suitable mammals include members of the orders Primates, Rodentia,Lagomorpha, Cetacea, Carnivora, Perissodactyla and Artiodactyla. Membersof the orders Perissodactyla and Artiodactyla are particularly preferredbecause of their similar biology and economic importance.

For example, Artiodactyla comprises approximately 150 living speciesdistributed through nine families: pigs (Suidae), peccaries(Tayassuidae), hippopotamuses (Hippopotamidae), camels (Camelidae),chevrotains (Tragulidae), giraffes and okapi (Giraffidae), deer(Cervidae), pronghorn (Antilocapridae), and cattle, sheep, goats andantelope (Bovidae). Many of these animals are used as feed animals invarious countries. More importantly, many of the economically importantanimals such as goats, sheep, cattle and pigs have very similar biologyand share high degrees of genomic homology.

The order Perissodactyla comprises horses and donkeys, which are botheconomically important and closely related. Indeed, it is well knownthat horses and donkeys interbreed.

As used herein, the term “effective amount” is meant an amount of acompound of the present invention effective to yield a desiredantimicrobial or radioprotective activity.

The specific “effective amount” will, obviously, vary with such factorsas the particular condition being treated, the physical condition of thesubject, the type of subject being treated, the duration of thetreatment, the nature of concurrent therapy (if any), and the specificformulations employed and the structure of the compound or itsderivatives.

The term “radiation damage” is used herein in its broadest sense andrefers to damage resulting from exposure to a radiation source, such as,ionising radiation. The term “ionising radiation” as used herein refersto photons having enough energy to ionise a bond, such as, α, β and γrays from radioactive nuclei and x-rays.

The term “cancer radiotherapy” is used herein in its broadest sense andinclude radiotherapy involving tumours which may be either benign ormalignant.

The primary application of the radioprotector of the present inventionis in cancer radiotherapy. Many of the normal tissues which are aproblem in radiotherapy such as the skin, oral mucosa, oesophagealmucosa, rectal mucosa, vaginal mucosa and bladder epithelium can beprotected by the radioprotectors of the present invention.

Outside the context of cancer radiotherapy, the radioprotectors of thepresent invention could be used prophylactly in high risk radiationsituations.

The compounds of the present invention may additionally be combined withother medicaments to provide an operative combination. It is intended toinclude any chemically compatible combination of pharmaceutically-activeagents, as long as the combination does not eliminate the activity ofthe compound of formula I or Ia. It will be appreciated that thecompound of the invention and the other medicament may be administeredseparately, sequentially or simultaneously.

Other medicaments which may be used when treating microbial infectionsinclude other anti-infective agents such as antibiotics.

When the compounds are used as radioprotectors the other medicaments mayinclude chemotherapeutic agents, for example, radiomimetic agents whichare cytotoxic agents that damage DNA in such a way that the lesionsproduced in DNA are similar to those resulting from ionising radiation.Examples of radiomimetic agents which cause DNA strand breaks includebleomycin, doxorubicin, adriamycin, SFU, neocarcinostatin, alkylatingagents and other agents that produce DNA adducts. It is anticipated thatthe radioprotectors of the present invention will protect DNA fromdamage by some of these agents, in the same way as they protect againstthe effects of ionising radiation. In clinical applications, it isunlikely that the radioprotector would be administered systemicallytogether with the chemotherapeutic agent, since this could compromisethe action of this agent on the tumour. However, there are circumstanceswhere topical application to problem tissues could be advantageous. Forexample, oral mucositis is problem side-effect for cytotoxic agents,such as, doxorubicin and administration of the present radioprotector asa mouth-wash before administration of the chemotherapeutic agent couldameliorate this side-effect without compromising the action of thisagent on a tumour not located in the oral cavity. Similarly, thegastrointestinal tract could be protected by oral administration, thelungs by aerosol inhalation or the bladder by intravesical delivery, forexample, via a catheter of the radioprotector. Hence a preferred methodin accordance with the present invention utilises the compound offormula I or Ia in conjunction with another medicament, such as, aradiomimetic agent.

The compounds of the invention may be conjugated to agents, for example,via the interactive group, which will specifically deliver them to adesired tumour site. Suitable agents may include antibodies or proteins,growth factors, for example, haemopoietic growth factor which willenable preferential radioprotection of haemopoietic stem cells to occurin the context of total body irradiation and bone marrowtransplantation.

There is also an ex vivo application of the conjugates of the compoundsof the invention in the context of bone marrow transplantation. Bonemarrow transplantation generally involves obtaining and storing bonemarrow samples from a subject in anticipation of a deterioration oftheir condition. A rather drastic form of chemotherapy (i.e. a highdose) is then administered. This chemotherapy is such that it wouldnormally be lethal due to the destruction of normal stem cells, but thesubject is rescued by the administration of their own haemopoietic stemcells. The problem with this procedure is that the initial sample ofstem cells is likely to be contaminated with tumour cells and variousprocedures are use therefore to purge the bone marrow preparations ofthe tumour cells. Radioprotectors conjugated to a haemopoietic growthfactor could be used in this context by being added to a suspension ofbone marrow cells. The suspension could then be irradiated in theexpectation that the normal bone marrow cells, but not the tumour cells,would be preferentially protected from the cell-killing effects of theradiation.

As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent or vehicle for delivering thecompound of formula I or Ia to the subject. The carrier may be liquid orsolid and is selected with the planned manner of administration in mind.Each carrier must be pharmaceutically “acceptable” in the sense of beingcompatible with other ingredients of the composition and non injuriousto the subject.

The compound of formula I or Ia may be administered orally, topically,or parenterally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.The term parenteral as used herein includes subcutaneous injections,aerosol for administration to lungs or nasal cavity, intravenous,intramuscular, intrathecal, intracranial, injection or infusiontechniques.

The present invention also provides suitable topical, oral andparenteral pharmaceutical formulations for use in the novel methods oftreatment of the present invention. The compounds of the presentinvention may be administered orally as tablets, aqueous or oilysuspensions, lozenges, troches, powders, granules, emulsions, capsules,syrups or elixirs. The composition for oral use may contain one or moreagents selected from the group of sweetening agents, flavouring agents,colouring agents and preserving agents in order to producepharmaceutically elegant and palatable preparations. Suitable sweetenersinclude sucrose, lactose, glucose, aspartame or saccharin. Suitabledisintegrating agents include corn starch, methylcellulose,polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.Suitable flavouring agents include peppermint oil, oil of wintergreen,cherry, orange or raspberry flavouring. Suitable preservatives includesodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methylparaben, propyl paraben or sodium bisulphite. Suitable lubricantsinclude magnesium stearate, stearic acid, sodium oleate, sodium chlorideor talc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate. The tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets.

These excipients may be, for example, (1) inert diluents, such ascalcium carbonate, lactose, calcium phosphate or sodium phosphate; (2)granulating and disintegrating agents, such as corn starch or alginicacid; (3) binding agents, such as starch, gelatin or acacia; and (4)lubricating agents, such as magnesium stearate, stearic acid or talc.These tablets may be uncoated or coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. Coating may also be performed using techniques described inthe U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

The compound of formula I or Ia as well as the pharmaceutically-activeagent useful in the method of the invention can be administered, for invivo application, parenterally by injection or by gradual perfusion overtime independently or together. Administration may be intravenously,intraarterial, intraperitoneally, intramuscularly, subcutaneously,intracavity, transdermally or infusion by, for example, osmotic pump.For in vitro studies the agents may be added or dissolved in anappropriate biologically acceptable solvent or buffer and added to acell or tissue.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents, growth factorsand inert gases and the like.

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a disease or sign orsymptom thereof, and/or may be therapeutic in terms of a partial orcomplete cure of a disease. “Treating” as used herein covers anytreatment of, or prevention of disease in a vertebrate, a mammal,particularly a human, and includes: (a) preventing the disease fromoccurring in a subject that may be predisposed to the disease, but hasnot yet been diagnosed as having it; (b) inhibiting the disease, i.e.,arresting its development; or (c) relieving or ameliorating the effectsof the disease, i.e., cause regression of the effects of the disease.

The invention includes various pharmaceutical compositions useful forameliorating disease. The pharmaceutical compositions according to oneembodiment of the invention are prepared by bringing a compound offormula I or Ia, analogues, derivatives or salts thereof, orcombinations of compound of formula I or Ia and one or morepharmaceutically-active agents into a form suitable for administrationto a subject using carriers, excipients and additives or auxiliaries.Frequently used carriers or auxiliaries include magnesium carbonate,titanium dioxide, lactose, mannitol and other sugars, talc, milkprotein, gelatin, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 20th ed. Williams & Williams (2000), the British NationalFormulary, 43^(rd) edition (British Medical Association and RoyalPharmaceutical Society of Great Britain, 2000), the contents of whichare hereby incorporated by reference. The pH and exact concentration ofthe various components of the pharmaceutical composition are adjustedaccording to routine skills in the art. See Goodman and Gilman's ThePharmacological Basis for Therapeutics (7th ed., 1985).

The pharmaceutical compositions are preferably prepared and administeredin dose units. Solid dose units may be tablets, capsules andsuppositories. For treatment of a subject, depending on activity of thecompound, manner of administration, nature and severity of the disorder,age and body weight of the subject, different daily doses can be used.Under certain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

The pharmaceutical compositions according to the invention may beadministered locally or systemically in a therapeutically effectivedose. Amounts effective for this use will, of course, depend on theseverity of the disease and the weight and general state of the subject.Typically, dosages used in vitro may provide useful guidance in theamounts useful for in situ administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of the microbial infections. Variousconsiderations are described, e.g., in Langer, Science, 249: 1527,(1990). Formulations for oral use may be in the form of hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin.They may also be in the form of soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium, such as peanut oil,liquid paraffin or olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspension. Suchexcipients may be (1) suspending agent such as sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2)dispersing or wetting agents which may be (a) naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The compound of formula I or Ia may also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

The compound of formula I or Ia may also be presented for use in theform of veterinary compositions, which may be prepared, for example, bymethods that are conventional in the art. Examples of such veterinarycompositions include those adapted for:

(a) oral administration, external application, for example drenches(e.g. aqueous or non-aqueous solutions or suspensions); tablets orboluses; powders, granules or pellets for admixture with feed stuffs;pastes for application to the tongue;

(b) parenteral administration for example by subcutaneous, intramuscularor intravenous injection, e.g. as a sterile solution or suspension; or(when appropriate) by intramammary injection where a suspension orsolution is introduced in the udder via the teat;

(c) topical applications, e.g. as a cream, ointment or spray applied tothe skin; or

(d) intravaginally, e.g. as a pessary, cream or foam.

Dosage levels of the compound of formula I or Ia of the presentinvention may be of the order of up to about 1 g per kilogram bodyweight. The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage will vary depending uponthe host treated and the particular mode of administration. For example,a formulation intended for oral administration to humans may contain upto about 1 g of an active compound with an appropriate and convenientamount of carrier material which may vary from about 5 to about 95percent of the total composition. Dosage unit forms will generallycontain between from about 5 mg to about 500 mg of active ingredient.

Optionally the compounds of the invention are administered in a divideddose schedule, such that there are at least two administrations in totalin the schedule. Administrations are given preferably at least every twohours for up to four hours or longer; for example the compound may beadministered every hour or every half hour. In one preferred embodiment,the divided-dose regimen comprises a second administration of thecompound of the invention after an interval from the firstadministration sufficiently long that the level of active compound inthe blood has decreased to approximately from 5-30% of the maximumplasma level reached after the first administration, so as to maintainan effective content of active agent in the blood. Optionally one ormore subsequent administrations may be given at a corresponding intervalfrom each preceding administration, preferably when the plasma level hasdecreased to approximately from 10-50% of the immediately-precedingmaximum.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of Log. No. survivors vs time (hours) for Candidaalbicans in Example 9; and

FIG. 2 is a graph of % parasitised blood cells vs hours of culture inExample 26 in which T=Trophozoites, R=Rings and T/S=Trophozoites orSchizonts.

EXAMPLES

The invention will now be described in detail by way of reference onlyto the following non-limiting examples and drawings.

Example 1 General Synthesis Methods

Benzdioxols are described in the literature (Perekalkin, 1982a). Thesynthesis of benzoimidazole and benzoxazole may also be carried outusing standard condensation methods 1 and 2 (Perekalkin, 1966, 1982b) asshown below.

in which X, Y,

and R₁ to R₇ are as defined in formula I above.

In Method 2, equimolecular quantities of benzaldehyde and nitroalkanewere mixed in an Erlenmayer flask and dissolved in equal volume ofalcohol. Fresh distillated ethylenediamine was added to the obtainedsolution in catalytical quantities (usually 1:10 in relation to aldehydeand nitroalkane) and was left in the dark at room temperature forseveral days (from 3 up to 10 days). During this time compoundcrystallised. After the cooling up to about 0° C., the crystals werefiltrated and washed with cold alcohol and then dried. When the yield issmall, the mother liquids can be joined together and evaporated inrotary evaporator. After cooling the additional quantity of impureproduct is obtained. The product was purified by dissolving in a minimalquantity of boiling alcohol. It was then treated with activated carbon,filtered hot and while the cooling was in progress, fine yellow needlescrystallised. The yield was about 80-85%, the compound beingchromatographically homogeneous.

The infrared spectra of the compounds obtained are in accordance withthose described in the literature (Hamlin and Weston, 1949; Knoevenageland Walter, 1904; Burton and Duffield, 1949).

The compounds were soluble in organic solvents such as ethanol, acetone,benzene, methanol, acetonitrile, chloroform and DMSO, but showed verypoor solubility in water (0.1%). When an alcoholic solution was added towater, a stable colloidal mixture was formed.

Example 2 Method for Preparing Compound (1)(3,4-methylenedioxy-β-methyl-β-nitrostyrene)

Compound (1) was prepared using Method 1 described in Example 1 above.The reaction scheme is shown below.

A mixture of 9.8 g of tetranitromethane (1 mole) and 10 cm³ of acetonewas cooled by ice and added dropwise to 8.1 g of distilled isosafrole (1mole) and 4.8 g of pyridine (1.2 mole) dissolved in 20 cm³ of acetone.The very first drops caused darkening of the reaction mixture and theliquid turned non-transparent and murky red when the entire portion oftetranitromethane was added. The smell of tetranitromethane disappearedquickly and in approximately 2 hours the dark red solution which hadturned transparent was poured into 100 cm³ of water in a stopperedbottle. The mixture was thoroughly shaken, covered with a layer of etherand a mixture of 6.7 cm³ of 33% solution of caustic potassium (1.03mole) and 50 cm³ of water was added in small portions. The mixture wasshaken after each addition and once the entire amount of alkali wasadded, the shaking was continued until the entire salt of pyridine andnitroform, which is present as a dark red oil, disappeared. The waterlayer was then separated and again extracted with ether. Combined etherextracts were first rinsed with water and then with water acidified withsulphuric acid and finally once again with pure water. Afterdistillation of the ether in the vacuum, a sediment of β-nitroisosafrolewas to be found in the form of yellow needles, which werere-crystallized from approximately 65 cm³ of alcohol. Compound (1) wasobtained with a melting point of 98° C. and a yield of 7 g. Once thesolvent had evaporated, another 0.5 g of Compound (1) was obtained. Thetotal product amounted to 72.5% of the theoretical yield.

Example 3 Alternative Method for Preparing Compound (1)(3,4-methylenedioxy-β-methyl-β-nitrostyrene)

Compound (1) was prepared using Method 2 described in Example 1 above.The reaction scheme is shown below.

900 gm piperonal was dissolved in 1000 cc alcohol with constant shakingand 450 ml nitroethane was added slowly followed by 10 ml ethyldiamine.After 17 hrs stirring, the mixture was placed in the dark at roomtemperature for 5-7 days. The resulting yellow crystals were filtratedin a Buchner funnel until dried and then washed twice with 150 mlalcohol. This yielded 1200 gm of Compound (1) with melting point of 95°C. After further crystallization from ethanol, 1000 gm of light yellowcrystals were obtained with a melting point of 98° C. (approx 80%yield).

Molecular formula C₁₀H₉NO₄, molecular weight—207.05

Physical and Chemical characteristics Form of state yellow crystalsSolubility profile <soluble in ethanol, acetone, benzene, methanol,acetonitrile, chloroform, DMSO - almost insoluble in water Melting point94-98° C. (when crystallized from 50% ethanol product had 96-98° C.) pH(in 50% v/v ethanol) approximately neutral Specific rotation opticallyinactive but has 2 stereoisomers Stability begins to darken above 200°C. Purity MS indicates impurities of molecular weight 303.4 & 331.4 tobe the major impuritiesIR Spectrum

-   1. Aromatic ring—above 3000 wave number & associate aromatics    1470-1630 region-   2. β-methylstyrene—additional groups over styrene 1442    aliphatic-C-+900-1000 peaks-   3. Nitrogroup at low wave number e.g. 747, 673 and β-nitrostyrene    has 1520.-   4. Aromatic Ether Group—1312 (1258) 1138, 1030 Nevertheless a    fingerprint of this compound is provided by the IR spectrum (q.v.).    This has been done on the recrystallised material in order to reduce    peaks due to contaminants.    IR Spectrum    Impurities of molecules weight 303.4 & 331.4    Confirmation of molecular weight of main species 207.1    NMR Spectrum

Hydrogen NMR (200 MHz) shows:

-   -   Aromatic ring with 3 remaining Hs, 3 Hs as part of a CH₃,        another attached to the side chain and 2 Hs as part of another        ring.

Carbon NMR (50 MHz) shows:

-   -   —CH₃, CH—, —CH₂ (as methylenedioxy)

Values of chemical shifts support the structure given and a likelihoodof the E-stereoisomer rather than the Z-stereoisomer favoured by thesynthesis used. A strong-withdrawing group (NO₂) is indicated.

UV/Visible Spectrum

Recrystallised material has peaks (broad) at 250-270 mm and 360-370 mmwith high absorbance below 210 mm.

Example 4 Process for Preparing Compound (2)

Compound (2) was prepared using Method 2 described in Example 1 above.The reaction scheme is shown below.

3,4-methylenedioxybenzaldehyde was condensed with nitromethane usingfresh distillated ethylenediamine NH₂—CH₂—CH₂—NH₂ as a catalyst. Thereaction was conducted in alcohol, darkness and at room temperature for5 days. The resultant crystals were separated by filtration and washedwith cold alcohol. After being dried in air, the yield was 80%,m.p.—158-159° C. and after re-crystallization the m.p. was 162-163° C.Compound (2) was non-soluble in water, soluble in acetone, alcohol,acetic acid and in a majority of organic solvents.

Example 5 Antibacterial Activity

In the experiments described herein, museum strains of pathogensobtained from the museum of the Microbiology chair of the MilitaryMedical Academy (designated by the index “M”) and strains selected frompathological material (designated by the index “B”), taken from patientsand having gone through no more than three laboratory passages wereused. For each type of pathogen the corresponding optimal nutrient mediawas used. For the impregnation method, compounds (1) and (2) were addedto solid nutrient media at doses from 0.03% to 2.0%. An agar diffusionassay analogous to the standard method of determining sensitivity toantibiotics was used.

The agar diffusion assay was performed as follows.

Meat peptone agar was prepared and impregnated with the test compoundsat concentrations from 0.01 to 2.0%. The medium was poured into Petridishes and allowed to set. Agar plugs of 10 mm diameter were cut out andplaced on the surface of Petri dishes containing the same mediumimmediately after they were inoculated with the microorganisms to beinvestigated (at least 6 plugs per culture). After one day of incubationat 37° C., the diameter of the zone of retardation of growth of theculture around the plugs was measured. The results were evaluated inaccordance with official standards of testing sensitivity toantibiotics; a diameter 20 mm corresponded to a stable culture, 21-28 mmto moderate stability and 29 mm to sensitivity.

In parallel to this, the sensitivity of pathogens to 15 antibiotics weretested according to the official protocol of the Russian SupervisoryAuthority for the Introduction of New Medicinal Substances and MedicalTechnology (disc method).

Several pathogen types and strains were used to show the limits ofsensitivity of the strains and types to the test substances, in order toevaluate their probable overall breadth of performance.

Table 1 below shows the results of experiments with Compound (1) at aconcentration of 1.0%, at which it suppressed the propagation of5×10⁵-5×10⁷ organisms/mL, and comparative results of sensitivityexperiments using the following 15 antibiotics:

1—penicillin,

2—ampicillin,

3—gentamycin,

4—carbenicillin,

5—kanamycin,

6—lincomycin,

7—levomicethin,

8—oxacillin,

9—polymixin,

10—rifampicin,

11—ristomycin,

12—streptomycin,

13—tetracycline,

14—erythromycin,

15—cephalosporin.

TABLE 1 Suppression of Growth (+/−) Compound Antibiotics Microorganism(1) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1. Conditional pathogenicenterobacteria gram- negative aerobic bacilli Pseudomonas aeruginosa + −− − − − − − ± − − ± − ± − − B-601 Escherichia coli M-17 + + + + + + − +− + − − ± + + + Escherichia coli B-683 + + + + + + − + − + − − ± + + +Escherichia coli B-65 + + + + + + + + − + − + ± + + + Enterobacteraerogenes + − − − − − − − − + − − − − − − B-679 Enterobacter aerogenes +− − − − − − − − + − − − − − − B-687 Citrobacter diversus + − + + − + − +− ± − − ± ± − + B-678 2. Pathogenic gram- negative bacteria Shigellaflexneri + − ± + ± ± − + − ± ± − ± ± − ± M-2A6907 Shigella sonneiB-720 + − ± − ± ± − ± − ± ± − ± ± − ± Salmonella typhimurium + − − − ± ±− − − ± ± − ± ± − ± M-727 Salmonella paratyphi + − ± − ± ± − ± − − − − ±± − ± M-16469 Acinetobacter B-681 + − − − − − − − − − + − − − − −Acinetobacter B-676 + − − − − − − − − − + − − − − − AcinetobacterB-677 + − − − − − − − − − + ± − − − − Alkaligenes sp. B-689 + − − + − +− + − − − − ± − − − Alkaligenes sp. B-667 + − − − − − − − − + + − − − −− 3. Gram-negative non- fermenting bacteria Yersinia + − − − − − − − − −− − − − − − pseudotuberculosis Klebsiella pneumoniae + − − − − − − − − −− − − − − − M-A21 Klebsiella pneumoniae + − − − − − − − − − − − − − − −M-248 4. Gram-negative aerobic bacilli Corynebacterium + − ± + − − − ± −− + + − + + + diptheriae B-670 Bacillus sp. B-575 + − − ± − ± + + − − ±± ± + ± − 5. Gram-positive aerobic cocci (Neisseria) Neisseriameningitidis + − − − − − − − − − − − − − − − M-6231 Neisseriameningitidis + − − − − − − − − − − − − − − − M-A72 6. Gram-positivecocci (Staphylococci) Staphylococci aureus + + − + + + − − + − + + + −− + M-12159 Staphylococci aureus + + − + + + − − − + + + + − − + M-209Staphylococci aureus + − − + + + − − + − + + + − − + B-685 Staphylococciaureus + − − + − − − + − − − − ± + + + B-674 Staphylococci + + + + + − +− + − + + − − − + epidermidis D-513 Staphylococci + + + − + − − − +− + + − − − − epidermidis BK-30 7. Gram-positive cocci (Streptococci)γ-haemolytic + + + − − − − − − − − + − − − − streptococcus B-672β-haemolytic + + + − + − − − − − + + − − − − streptococcus B-624Remarks: + sensitive strains ± moderately sensitive strains − stablestrains

-   -   Table 2 below shows the results of experiments on sensitivity of        some microorganisms to Compound (1) using the agar diffusion        method described above.

TABLE 2 Diameter of zone of Growth inhibition (mm) Concentration in discof Compound (1) (%) Microorganism 2.0 1.0 0.1 γ-haemolytic 35# 34# 32#streptococcus B-672 β-haemolytic 31# 32# 31# streptococcus B-624Staphylococci aureus 39# 37# 35# B-685 Staphylococci 38# 34# 32#epidermidis B-513 Shigella flexneri M- 36# 33# 31# 2A6907 Shigellasonnei B- 39# 35# 30# 720 Salmonella 32# 30# 24# typhimurium M-727Salmonella paratyphi 36# 30# 28# M-16469 Acinetobacter B-681 38# 35# 32#Alkaligenes sp. 40# 38# 36# B-689 Enterobacter 33# 30# 25# agglomeransB-679 Corynebacterium B- 40# 35# 31# 670 Bacillus sp. B-575 38# 34# 29 Remarks: # sensitive + moderately sensitive − stable

Table 3 shows the results of experiments of sensitivity of pathogenicfungi to compound (1).

TABLE 3 Number of microbes in the presence of Compound (1) at Dosage ofconcentration (%) Fungi Inoculation Result 2.0 0.5 0.1 Candida 5 × 10⁴Growth  0  0  0 albicans B-45 Retardation 5 × 10⁴ 5 × 10⁶ 5 × 10⁶Candida 5 × 10⁴ Growth  0  0  0 albicans M-3 Retardation 5 × 10⁶ 5 × 10⁶5 × 10⁶ Trichophyton 10² Growth  0  0  0 Retardation 10⁷ 10⁷ 10⁷Geotrichum 10⁵ Growth  0  0  0 M-158 Retardation 10⁵ 10⁵ 10⁴ Torula 10⁵Growth  0  0  0 histolytica Retardation 10⁵ 10⁵ 10⁵ (Cryptococcus)

Example 6 Antimicrobial Tests

The antimicrobial activity of Compound (1) was tested, using the agardiffusion method.

Due to the very low solubility of the compounds, the studies could notbe carried out in the liquid phase. For this reason an initial motherimpregnate, containing 0.5% of test compound, was prepared on meatpeptone agar. From this mother impregnate, regenerated to the liquidstate in a water bath, serial double dilutions were prepared by additionof the agar base. The dilutions thus obtained, containing the compoundsin concentrations of 0.5%, 0.25%, 0.12%, 0.06%, 0.03%, 0.015% werepoured into Petri dishes, on which 6 bacterial and 2 fungal testcultures were inoculated.

The following test cultures were used:

1) Staphylococcus aureus strain 674, isolated from a patient, sensitiveto gentamycin, oxacillin, tetracycline, erythromycin and cephalothin,slightly sensitive to streptomycin.

2) Enterococcus faecalis museum strain, sensitive to ampicillin,rifampicin, and streptomycin.

3) Klebsiella pneumoniae strain 312, isolated from a patient, sensitiveto gentamycin and polymyxin.

4) Salmonella typhimurium museum strain 727, sensitive to ampicillin,gentamycin, carbenicillin, canamicillin, polymyxin, and cephalothin.

5) Acinetobacter strain 681, isolated from a patient, slightly sensitiveto polymyxin.

6) Pseudonomas aeruginosa strain 328, isolated from a patient, slightlysensitive to polymyxin.

7) Trichophyton interdigitale.

8) Candida albicans.

Each of the 8 test cultures was sown on sterile meat peptone agar in aPetri dish, and then a standard plug of agar, impregnated with one ofthe 9 compounds at a concentration of 0.5%, was placed on the agarsurface. The retardation zone was measured around the plug after 24 hand 48 h growth at 37° C. For the fungal cultures the results wereassessed after 7-10 days of incubation at 30° C.

The results of the action of the compound on the impregnated agar aresummarised in Table 4.

TABLE 4 Minimal % Compound (1) giving complete inhibition of %inhibition test culture by antibiotic S. aureus 0.015 5.5 Enterococcusfaecalis 0.015 3 Klebsiella pneumoniae 0.12 2 Salmonella typhimurium 0.55 Acinotobacter 0.12 1 Pseudomonas — 1.5 Trichophyton 0.25 — Candida 0.5— Activity Index 0.4 —

Table 5 below shows the results of the agar diffusion experiments.

TABLE 5 Results of growth retardation by the compounds in the diffusionsin the agar experiment Size of zone of retardation of Test culture thegrowth by Compound (1) Staphylococcus aureus 6.5 Enterococcus fecalis 6Klebsiella pneumonia 5 Salmonella typhimurium 7 Acinetobacteria 6.5Pseudonomas 0 Average zone of retardation 5.17In equal concentration Compound (1) inhibits the growth ofampicillin-resistant Staphylococcus and the growth of Enterococcus whichis sensitive to this antibiotic. Similar differences can be observedwith other pathogens and other preparations.Anti TB Effects of Compound (1)

Anti TB effect was checked by standard method of serial double dilutivein synthetic liquid medium (SOTON) with 10% normal equine serum. Thesolution was prepared in Tween 80.

Test culture was Mycobact tub. H.37RV sensitive to anti TB medication.

Mycobacterial suspension (density 5×10⁷ cells/ml) was spread onto aspecial liquid medium (Vischnevsky, B.I.)

Results were calculated for 10-14 days incubation at 37° C. MIC (totallyinhibiting M. tuberculosis)

The results are shown in Table 6 below.

TABLE 6 Minimal Inhibitory Concentration (μg/ml) Compound (1) 6.25Isoniazid 0.02-0.1  Rifampicin 0.01-0.02 Ethambutol 1.0-2.5 Streptomycin0.5 

Only Compound (1) had MIC close to Ethambutol/Isoniazid MIC.

Example 7 Anti-Protozoal Activity

The effect of the Compounds (1) and (2) on trichomonas was alsoinvestigated. Trichomonas vaginalis isolated from patients was used. Thetrichomonads were cultured at pH 5.8-6.5 and 37° C. in medium 199containing 5.0% native foetal calf serum, carbohydrates, and antibioticsto suppress the accompanying flora. Vaseline was applied to the surfaceof the medium in the culture tube. The experimental specimens containedthe test compounds at a concentration of 0.3%.

7 specimens were investigated, containing motile forms of the parasitein a quantity of 5-8 cells in 1.0 cm³. In the control, parasites werecultured successfully over 3-4 passages (each passage 5-6 days). Incontrast, culture of motile forms in medium containing the testcompounds was unsuccessful in every case. After only one passage in thepresence of the test compounds, motile forms did not propagate.

Example 8 Anti-Bacterial Activity In Vivo

The therapeutic and prophylactic effect of the substances was determinedin experiments in vivo on mice, infected intraperitoneally orintranasally with Corynebacterium paratuberculosis.

Compounds (1) and (2) were administered intraperitoneally,intramuscularly and orally at a dose of <20% LD₅₀/0.2 at differentperiods from the day of infection, ie 2 and 1 days before the infection(schedules 2, 1), on the day of infection (schedule 0) and 1, 2, 3 etc.days after the infection (schedules +1, +2, +3, etc).

The daily mortality rate was measured, their cumulative variations werecalculated and based on this the results of the performance of thepreparation was determined by the formulaAI=[(B−A):B]×100where

AI=activity index of the preparation (%),

A=cumulative mortality in the experimental group,

B=cumulative mortality in the control group.

The results are shown in Table 7 below and indicate that Compounds (1)and (2) tested showed therapeutic and prophylactic activity in miceinfected with Corynebacterium paratuberculosis.

For intramuscular administration, the clinical prophylactic performancein the form of reduced mortality of animals was 52.53%. The clinicalperformance for salmonellosis varied within the limits of 50.0-20.0%.For pseudotuberculosis the prophylactic performance was 50.0%.

TABLE 7 Order of activity of compounds as assessed by AI Incubationperiod Cumulative days mortality Pathogen and method of Schedule ofintroduction of (test/control) (test/control) Compound infection thepreparation test contr. test contr. Activity index % 1 Salmonellaintraperitoneally −1, 0, +1, 0 0 47 99 +52.53 +2, +3 200 mg/kgintramuscularly 2 Salmonella intranasally +2, +3 1 0 15 30 +50.00 200mg/kg intramuscularly

Example 9 Radiation Protective Activity

The radiation protective performance of the Compounds (1) and (2)investigated was tested on male white mice weighing 18-20 g.

Dosages: 2, 4, 6, 8, 10, 15, 20 Gr (1 hR=100 Röntgen).

Period of observation=25 days.

Method of reporting: dynamic mortality, calculation of cumulativemortality and factual changes of dosage (FCD).

Schedule of introduction: 50 mg/kg at 0.2 mL in mice.

Group 1—control,

Group 2—2 and 1 days up to irradiation.

Table 8 shows the results of the experiments on radiation performance ofthe compounds investigated.

TABLE 8 Cumulative mortality in groups % Dosage R (hR*) controlprophylactic 2 2.9 0 4 3.6 0 6 13.1 3.8 8 21.1 9.5 10  43.8 13.3 15 66.7 45.5 20  94.1 83.3 FCD 1.39 + 1.48 LD₅₀ 10.73 hR 15.88 hR Remarks:*1 hR = 100 Rontgen

The results show that for all irradiation dosages prophylacticadministration of the test compounds considerably decreases themortality of irradiated animals in comparison with the control group.FCD (LD_(50 contr.))=1.39-1.43, which shows a high radiation protectiveeffect of the compounds indicated, the performances of which do notyield to the reported media.

Example 10 Treatment of Infected Wounds

Human volunteers suffering from skin wounds infected by Streptococcusand Staphylococcus were treated with 0.1% Compounds (1) and (2) in anointment base of vaseline, sheep fat and sulfoxide. Application of 0.1%ointment for 3 days cleared the wound completely of pus, with subsequenthealing of the wound.

In 3 cases extensive damage to the skin caused by fungal infection wastreated; the type of the fungus was not identified. The damaged area wassmeared twice per day with 0.1% ointment. The skin was cleared of fungalgrowth within a week.

Example 11 Pharmacokinetics

While the pharmacokinetics of the compounds of the invention have notbeen investigated in detail, in experiments carried out in mice to whichCompounds (1) and (2) were administered intraperitonally andintragastrically, it has been established that the compound will remainin a biologically active concentration in the blood for longer than 24h.

Example 12 Toxicology

The average lethal dose for mice when administered intragastrically was1500 mg/kg body weight; when administered intraperitonally the averagelethal dose was 575 mg/kg body weight. Thus the compounds of theinvention have low toxicity.

Example 13 Antimicrobial Activity and Solubility of Compound (1)

Compound (1) is relatively insoluble in water but is soluble in 10% DMSOat 1 mg/mL (0.1%), 10% ethanol at 2 mg/ml and 10% acetone at 2 mg/mL.

The highest concentration testable is either 512 μg/mL at 5% solvent or256 μg/mL at 2.5% solvent. It does not require specific chemicalneutralization, dilution being sufficient to neutralize residualactivity in microbicidal testing.

DMSO was selected as the solvent for testing because it has the lowesttoxicity against test strains.

Compound (1) was at least 8 times more active against E. coli whenformulated in ethanol, giving an MIC of 128 μg/mL (0.06% ETON) comparedto >512 μg/mL (2.5% DMSO). Ethanol is toxic to E. coli at concentrations>2.5%.

Example 14 Antibacterial Activity of Compound (1)

NCCLS-USA Standard Method—Broth microdilution (or macrodilution)(Mueller-Hinton).

Inoculum 1-4×10⁴ cfu (or −4×10⁵ cfu). Ciprofloxacin test control.Chlorhexidine values added for disinfectant and antiseptic activitycomparison.

Minimum inhibitory concentration (MIC) and minimum bactericidalconcentration (MBC) as 3 log reduction (99.9% kill) at 35° C., 24 hours,aerobically (unless otherwise indicated). 48 h titres were notsignificantly different.

Results are shown in Table 9.

Summary of Results

Compound (1) is a relatively broad spectrum antibacterial agent withbactericidal activity within an acceptable concentration range in vitrofor a representative selection of Gram positive and Gram negativebacteria. Compound (1) is broadly effective against aerobic Grampositive and Gram negative cocci and aerobic Gram positive rods ofclinical significance.

Infections with Resistant Gram Positive Cocci

Clinical isolates (multiple antibiotic resistance) of S. aureus and E.faecalis were as susceptible as the standard strains. Although notactive at the low concentrations cf current treatment drugs, there maybe a potential use for Compound (1) as a treatment for multiplyresistant staphylococci and enterococci not responding to the currentdrugs of choice.

Anaerobic Infections

Compound (1) is active against clinically significant anaerobes,Clostridium perfringens, Clostridium difficile and Bacteroides fragilis.C. difficile causes enterocolitis in hospitalized patients and iscurrently treated with vancomycin as the drug of choice. Induction ofresistance is a potential problem with vancomycin. There could be amarket for an oral drug for C. difficile enterocolitis as an alternativeto vancomycin.

Anaerobic infections are generally mixed infections of one or moreanaerobes with facultative bacteria, usually enteric Gram negative rods.The most common anaerobic pathogens are Clostridium difficile andBacteroides fragilis. Current treatment with metronidazole incombination with other antibacterial drugs is generally efficacious.Given Compound (1)'s broad spectrum against both aerobic and anaerobicbacteria this could possibly treat these infections.

Enteric Infections

Compound (1) is very active against Campylobacter jejuni. Campylobacteris currently the greatest cause of enteric infections worldwide and isoften treated because of its severity in some patients and the tendencyfor infection to predispose to development of Guillain-Barre syndrome, aserious CNS disease.

The relative resistance of enteric Gram negative bacteria could be afunction of solubility and ability of Compound (1) to penetrate cells.The successful treatment of recalcitrant enteric infections and thesuccessful treatment of Salmonella infection in animals has been shown.

Vulvo-Vaginitis

Compound (1) is active against Neisseria gonorrhoeae. Vulvo-vaginitis iscaused by Candida albicans, N. gonorrhoeae, Chlamydia trachomatis andTrichomonas vaginalis (singly, not as co-infections). Compound (1) isactive against two of these agents.

Formulating Compound (1) for greater solubility (and therefore probablygreater absorption) could improve both its activity and its distributionin vivo.

TABLE 9 MIC/MBC (μg/mL) for Compound (1), ciprofloxacin andchlorhexidine against a range of bacteria of clinical significanceCompound (1) Ciprofloxacin Chlorhexidine Bacterial strain MIC MMC MICMMC MIC MMC Gram positive Staphylococcus aureus ATCC 29213 16 16 0.250.25 2 8 S. aureus - clinical isolate 1 16 16 S. aureus - clinicalisolate 2 16 16 Enterococcus faecalis ATCC 29212 32 32 0.5 0.5 E.faecalis - clinical isolate 1 32 32 1 1 E. faecalis - clinical isolate 232 32 Streptococcus pyogenes 16.8 16.8 Streptococcus pneumoniae ATCC49619 16 32 Bacillus subtilis RMIT 16 16 Corynebacterium xerosis RMIT32.16 32.16 Clostridium perfringens (48 h) 16 32 Clostridium difficile 4Gram negative Moraxella catarrhalis RMIT 32 32 Neisseria gonorrhoeae WHOStrain VII 2 2 Haemophilus influenzae 0.125 0.125 0.006 0.006Bacteroides fragilis (48 h) 16 32 Campylobacter spp. (48 h) 128 128Campylobacter jejuni RMIT FF3 (48 h) 2 2 Acinetobacter calcoaceticusRMIT 128 256 Proteus vulgaris RMIT 128 256 6 128 Proteus mirabilis256 >512 Klebsiella oxytoca 128 256 Klebsiella pneumoniae 512 >512Salmonella Typhimurium 256 >512 Escherichia coli ATCC 25922 >512 >5120.02 0.02 2 4 Pseudomonas aeruginosa ATCC 27853 >512 >512 0.25 0.25 3264 Serratia marcescens RMIT >512 >512 16 32 Enterobacter aerogenes 512512

Example 15 Antifungal Activity of Compound (1)

NCCLS-USA Broth macrodilution method (RPMI medium).

Inoculum ˜5×10⁴ hyphal fragments/mL (haemocytometer). Miconazolecontrol.

Minimum inhibitory concentration (MIC) and minimum fungicidalconcentration (MFC 2 log reduction—99% kill) at 30° C., aerobically, 2,7 and 10 days for yeasts and 4, 7 or 14 days for filamentous fungi.

Results are shown in Table 10.

Summary of Results

Compound (1) is fungicidal at relatively low concentrations against abroad range of clinically significant yeasts and filamentous fungi(Table 10).

Dermatophyte Infections

Compound (1) shows good activity against 3 major causes of skin, hairand nail infections in humans and animals. Superficial fungal infectionsare the most common fungal infections worldwide. Treatment is prolonged,over months (and years for nail infections). Superficial treatments withantifungal lotions and creams is only partially effective. Currenttreatments, although generally low cost, have poor efficacy and frequentrelapse rates. Oral systemic agents (terbinafine and itraconazole) arepreferred for superficial infections in compromised patients and nailinfections.

Systemic Infections

Serious fungal infections in compromised patients have increasedworldwide in prevalence and severity. Fungal infections are generallylong term with a high therapeutic failure rate, frequent relapse anddevelopment of resistance by fungi. Candidiasis (Candida albicans) andaspergillosis (Aspergillus fumigatus) are the major fungal infections.Severe, invasive infections have a high mortality rate. Very feweffective drugs are available (cf antibacterial drugs). Long termtherapy makes safety and failure to induce resistance importantconsiderations. Amphotericin B is the main drug of choice for manyserious mycoses. It is fungicidal, with poor solubility and lowbioavailability and is limited by toxicity and delivery problems andhigh therapy failure. The azoles and triazoles are fungistatic drugs (egfluconazole and itraconazole) which have low toxicity, goodpharmacokinetic characteristics but are often ineffective due todevelopment of resistance on long term therapy.

Compound (1) has a broad spectrum, is fungicidal and has failed toinduce resistance in C. albicans and A. fumigatus (see below).

TABLE 10 MIC/MFC (μg/mL) for Compound (1) and miconazole againstclinically significant fungi Compound (1) Miconazole Fungi MIC MMC MICMMC Dermatophytes (7 day) Trichophyton rubrum 1 256 1 64 Epidermophytonfloccosum 0.5 16 0.25 0.25 Microsporum gypseum 1 8 4 64 Yeasts (4 day)Candida albicans 8 8 4 8 Rhodotorula rubra 8 8 8 32 Filamentous fungi (7day) Fusarium graminearum 4 8 32 32 Rhizopus stolonifer 4 16 Aspergillusfumigatus 8 32 Penicillium chrysogenum 1 2

Example 16 Sporicidal Activity of Compound (1)

Bacillus subtilis endospores

Aspergillus fumigatus asexual exospores

Compound (1) in water+Tween 20 was tested for sporicidal activity up to24 hours.

Compound (1) 512 μg/mL did not kill B. subtilis endospores in 24 hours.

Compound (1) 512 μg/mL killed A. fumigatus exospores at 24 hours but not6 hours.

Inhalation of Aspergillus spores is the major mechanism of transmission.Activity against spores could be significant in prophylaxis ofcompromised individuals. Since the spore must germinate to infect,however, ultimately it is activity against vegetative forms of fungithat determine efficacy.

Example 17 Development of Resistance of Compound (1)

Bacterial and fungal strains were exposed to Compound (1) insub-inhibitory concentrations continuously for 12 weeks and monitoredfor a rise in MIC indicating development of resistance mechanisms. Aheavy and variable inoculum is used for weekly subculture so inhibitoryconcentrations each week vary. The standardized MIC is measured at thebeginning and end of exposure. A greater than 4-fold variation ofstandardized MIC is indicative of increased resistance, or a risingtrend in weekly MIC. The genera selected are known to develop resistancereadily to many antibiotics and to be a major clinical problem.

Bacterial species and C. albicans, known to develop resistance to manycurrent drugs, did not develop resistance to Compound (1) after 12 weekscontinuous exposure (Table 11). Strains were scanned for abnormalmicroscopic and macroscopic changes. Proteus vulgaris lost the abilityto swarm, indicating an effect on flagella. Other strains appearednormal. Tests are not yet complete for R. rubra and 3 moulds. Failure toinduce the development of resistance in these strains is a significantattribute of Compound (1).

TABLE 11 Increase in MIC (μg/mL) of bacterial and fungal strains after12 weeks continuous exposure to sub-inhibitory concentrations ofCompound (1) in MHB Standard 7 day MIC μg/mL MIC μg/mL no rising trendTest strains Initial Final Range to Week 12 Staphylococcus aureus 16 1632-128 (MRSA clin isolate) Enterococcus faecalis 32 32 64-128 (MR clinisolate) Klebsiella oxytoca 128 256 256-512  Proteus vulgaris 128 12864-256 Candida albicans 4 4 16-64  Rhodotorula rubra 8 8-32 to Wk 5Aspergillus fumigatus 8 8-32 to Wk 5 Rhizopus stolonifer 4 4-32 to Wk 5Fusarium graminearum 4 4-16 to Wk 5

Example 18 Antibacterial Activity of Compound (1) in the Presence ofBlood

The activity of Compound (1) and ciprofloxacin was determined in thepresence of plasma and whole blood (horse), by macrodilution method inMueller Hinton broth to 48 h.

Compound (1) appeared to be relatively unaffected by the presence of 10%plasma and to be more active in the presence of 5% whole blood. Theslightly improved inhibitory activity of drugs in the presence of bloodsometimes occurs with antibacterial agents and is probably due tonatural antibacterial factors present in blood. Further increasing theconcentration of plasma and blood reduced the bactericidal activity ofCompound (1) against S. aureus as shown in Table 13. Ciprofloxacinshowed respectively a 4-fold and 2-fold decrease in activity against Saureus in the presence of 10% plasma and whole blood.

TABLE 12 Activity of Compound (1) in the presence of human whole bloodand plasma μg/mL — 10% plasma 5% blood MIC MIC MMC MIC MMC S. aureus 1616 256 16 256 B. subtilis 16 8 8 8 16 A. calcoaceticus 128 128 128 64256 M. catarrhalis 32 8 8 4 8

Example 19 Compound (1) Binding to Plasma Proteins

The MIC of Compound (1) was determined in increasing concentrations ofplasma. Compound (1) has been shown to bind to human serum albumin andto agarose. Serum binding is significant in drug distribution andbioavailability.

The MIC of Compound (1) is significantly increased with increasingplasma concentrations. Bactericidal activity is much more affected thaninhibitory activity.

Bioavailability of Compound (1) is significantly decreased in thepresence of plasma proteins (Table 13). Compound (1) is reversibly boundto proteins.

TABLE 13 MIC/MMC (μg/mL) for Compound (1) in MHB in the presence ofincreasing concentrations of plasma Streptococcus % plasma in S. aureuspyogenes MHB MIC MIC 0 4 4 1 4 4 2.5 8 8 5 8 8 10 16 16 20 32 32 50 12864 100 128 256

Example 20 Rate of Kill

Test strains were inoculated into Compound (1) solutions in water andsampled immediately and at 1, 2, 4, 6 and 9 hours. Survivors wereestimated by viable counts on MHA (35° C., 48 h).

Measure of kill: reduction in viable count (log) expressed as logreduction factors.

(e.g., a I log reduction=90% kill, 2 log=99% kill, 3 log=99.99% killetc.)

Compound (1) showed rapid kill only against Candida albicans, withgreater than a 99.999% reduction within 2 hours at 512 μg/mL and within4 hours at 256 μg/mL as shown in FIG. 1.

Rate of kill was much slower against bacteria. The kill rate at 512μg/mL was 99.99% within 2 hours for B. subtilis and 99.99% within 9hours for S. aureus.

Ciprofloxacin was not tested.

TABLE 14 Log reduction factors for Compound (1) over 6 or 9 hours Logreduction in viable count at time (h) Compound (1) μg/mL 512 256 128Staphylococcus aureus   4 in 9 h 0.5 in 6 h Enterococcus faecalis 0.6 at9 h Bacillus subtilis   4 in 2 h 3.8 in 2 h Klebsiella oxytoca 0 0Proteus vulgaris   1 in 6 h 0.5 in 6 h Acinetobacter calcoaceticus   1in 6 h   1 in 6 h Candida albicans 5.5 at 2 h   6 in 4 h   2 in 6 h

Example 21 Dosing Range Test of Compound (1) in Rat

The aim of this example was to establish absorption and blood levels ofCompound (1) in the rat after a single dose oral administration.

Test Protocol

Sprague-Dawley rats (6 w/o, delivered 30 Jan. 2001) were acclimatisedfor 6 days in the Animal Facility under standardized environmentalconditions (22° C.±3° C., rel hum 30-70%, artificial light, 12 hlight/12 h dark). Rats were fed a conventional laboratory diet with foodand water ad lib and caged 5 rats per cage.

Test Substance

Compound (1) was prepared as an aqueous suspension in sterile LPW. Athigher concentrations the suspension was sonicated to reduce particlesize sufficiently to pass through the gavage needle.

Compound (1) was tested at 1250, 1000, 500 and 100 mg/kg.

Test Method

Rats were randomly assigned to treatment groups, identified by numberingon tails. Doses were tested sequentially from the lowest dose.

Group A  100 mg/kg  5/2/01 B  500  8/2/01 (not fasted) C  500 12/2/01(fasted) D 1000 14/2/01 E 1250 21/2/01

Compound (1) suspensions and the water control were administered atapprox 100 mL/kgbw, in a single. One control and five treatment ratswere weighed immediately before each dose administration, the dosevolume calculated and the dose delivered by gavage (22 gauge stainlesssteel, smooth-balled end attached to a syringe).

Approximately 100-200 μL of blood (microfuge tube) was removed from thetail at 4 and 8 hours. Tails were prewarmed using a heat lamp andsnipped at the tip with a large scalpel. Blood was massaged into amicrofuge tube. Twenty four hour blood samples were not attemptedbecause of the difficulty of snipping scarred tails and the distresscaused to rats.

Blood was allowed to clot, centrifuged in a microfuge for 3 minutes(speed 14) and the serum separated and stored at −20° C.

Animals were observed twice daily for 7 days and all observationsrecorded individually for each animal. Animals were not weighed afterthe initial weighing. Sacrifice and necropsy was performed at 7 days.

Animals were euthanized by carbon dioxide.

Gross pathology was recorded and samples of heart, lung, liver, kidney,stomach, spleen, duodenum and colon removed (10% formalin) forhistology.

Example 22 Blood Levels of Compound (1)

Bioassays

Bioassay for Compound (1) levels in blood was not possible because ofthe interference due to strong binding of Compound (1) to blood proteinsand to agar.

Agar Diffusion

An agar diffusion assay for Compound (1) was not possible becauseCompound (1) bound so strongly to agar that no zones of inhibition wereproduced at any concentration from 1 to 512 μg/mL with susceptiblestrains of S. aureus or Streptococcus pyogenes. Both well diffusion anddisk diffusion assays were attempted.

Broth Dilution

Dilution of serum in MHB and testing with a low inoculum of S. pyogenes(inhibited at 1 μg/mL of Compound (1) in MHB) was not possible becausestrong binding to plasma at high concentrations caused a significantprozone.

Assay by UV Spectroscopy

There was insufficient serum for assay of individual rat samples.

Samples for treatment groups and for controls were thus pooled and amean level of Compound (1) for each treatment group determined.

Test Method

Compound (1) was extracted (×2) from serum by toluene and absorbancemeasured at 370 nm (Hitachi U2000). A spiked control using 100 μg/mLCompound (1) in 50% methanol/water (V/V) and untreated controls werealso assayed.

Blood Levels

Absorption of Compound (1) from the gastrointestinal tract is very low,approximately 2% of the oral dose reaching the blood. Blood levelsincreased with dose level. Eight-hour levels were generally higher than4-hour levels. The small difference between 4 and 8 hour levels suggestsa slow absorption.

TABLE 15 Blood level of Compound (1) dose Sample time Compound (1) μg/mL(mg/kg) (hours) Mean  500 fed 4 8 8 17  500 fasted 4 12 8 14 1000 fasted4 26 8 21 1250 4 + 8 h 27

Example 23 Antimicrobial Activity Spectrum of Compound (1)

Antifungal Activity

Filamentous Fungi

NCCLS-USA Broth Macrodilution method (RPMI medium)—draft. Inoculum1-4×10⁵ cfu. Miconazole control. This test was used for initial activityspectrum evaluations.

The MIC and MFC of filamentous fungi tested previously with theNCCLS-USA Broth Macrodilution Method were repeated using the newproposed standard microdilution method for testing fungi M38-PNCCLS-USA.

Results are of 2 or 3 replicates on different days.

Results were not significantly different from results obtained with theolder method for all fungi previously tested. Amphotericin B wassubstituted for Miconazole as control for some tests.

Yeasts

NCCLS—M27-A Method for broth macro dilution antifungal susceptibilitytesting of yeasts; approved standard.

M38-P microdilution method for filmentous fungi also used.

Minimum inhibitory concentration (MIC) and minimum fungicidalconcentration (MFC—2 log reduction—99% kill) at 35° C., 48 hours.Results of 2 or 3 replicates on different days for each method. Resultswere not significantly different for the two methods. M38P only reportedfor yeasts and filamentous fungi.

TABLE 16 MIC/MFC (μg/mL) for Compound (1) and Amphotericin B againstclinically significant fungi - M38 - P method Compound (1) AmphotericinB Miconazole MIC MMC MIC MMC MIC MMC Yeasts (24 h, 35° C.) Candidaalbicans 8 8 0.25₁ 0.25 C. guillermondii RMIT 176 2 2 0.03 0.06 1 1 C.krusei RMIT 177 4 4 0.5₂ 0.5 2 2 C. parapsilosis RMIT 178 2 2 0.25₃ 0.250.5 0.5 C. tropicalis RMIT 181 4 4 0.25 0.5 1 2 C. glabrate RMIT 157 2 20.5 1 0.25 0.5 Cryptococcus neoformans 1 0.5 4 Filamentous fungi (48 h,35° C.) Aspergillus fumigatus 8 32 1 8 A. niger 8 16 2 2 A. flavus 16 328 8 Fusarium graminearum 4 8 4 8 F. chlamydosporum 8 8 2 2 Rhizopusstolonifer 4 16 >16 R. oryzae 64 64 4 4 Rhizomucor pusillus 4 8 1 1Paecilomyces variotii 1 2 1 1 Dematiaceous fungi Fonsecaea pedrosoi 2 28 16 Phialophora verrucosa 16 32 2 4 Pseudoallescheria boydii 2 4 4 16Dermatophytes Trichophyton rubrum 1 256 Epidermophyton floccosum 0.5 16Microsporum gypseum 1 8 Permitted range for Amphotericin B control0.25-1 μg/mL. 0.5-2.0 μg/mL 0.25-1 μg/mLBacteriaMethods

NCCLS-M7-A5 Standard Method—Broth microdilution (Mueller-Hinton).Inoculum 1-4×10⁴ cfu. Ciprofloxacin test control. Chlorhexidine andcetyl trimethyl ammonium bromide (CTAB) for disinfectant and antisepticactivity comparison.

NCCLS-M7-A5 Standard Method—macrodilution (Mueller Hinton±specifiedenrichments) was also used. Minimum inhibitory concentration (MIC) andminimum bactericidal concentration (MBC) as 3 log reduction (99.9% kill)at 35° C., 24 hours, aerobically. 48 h titres were not significantlydifferent and are not reported.

Micro and macro dilution methods did not give significantly differentMIC/MMC.

TABLE 17 All tests performed as 2 or 3 replicates on different days.μg/mL Compound (1) Ciprofloxacin Chlorhexidine CTAB Bacterial strain MICMMC MIC MMC MIC MMC MIC MMC Gram positive Staphylococcus aureus ATCC29213 16 16 0.25 0.25 2 8 16 32 S. aureus - clinical isolate 1 16 16 S.aureus - clinical isolate 2 16 16 Enterococcus faecalis ATCC 29212 32 320.5 0.5 E. faecalis - clinical isolate 1 32 32 E. faecalis - clinicalisolate 2 32 32 Streptococcus pyogenes RMIT 16 16 Streptococcuspneumoniae 2 2 Bacillus subtilis RMIT 16 16 Corynebacterium xerosis RMIT32 32 Gram negative Moraxella catarrhalis RMIT 32 32 Neisseriagonorrhoeae 2 2 Haemophilus influenzae 0.125 0.125 Acinetobactercalcoaceticus RMIT 128 256 Proteus vulgaris RMIT 128 128 6 128 128 256Proteus mirabilis 256 >512 Enterobacter aerogenes 512 512 Klebsiellaoxytoca 128 256 Klebsiella pneumoniae 512 >512 Escherichia coli ATCC25922 >512 >512 0.02 0.02 2 4 16 16 Pseudomonas aeruginosa ATCC27853 >512 >512 0.25 0.25 32 64 512 >512 Serratia marcescensRMIT >512 >512 16 32 128 128 Bacteroides fragilis 16 32Campylobacter

Compound (1) was tested against a range of clinical strains ofCampylobacter spp. isolated from humans.

TABLE 18 MIC/MMC (μg/mL) of Compound (1) by NCCLS - M7 A5 macro dilutiontest, 42° C., 48 h, microaerophilic incubation MIC MMC Campylobacterjejuni 54/1-2 2 2 C. jejuni 541-3 2 2 C. coli 54/2 4 4 C. foetus 54/3 22 C. hyointestinalis 54/4 2 2 C. sputorium 54/5 2 2 C. laniolis 54/6 2 2

Campylobacter is the most common cause of gastroenteritis infectionworldwide (bloody diarrhoea, abdominal pain, vomiting, headache, fever,lasting about 1 week). Sequelae are arthritis and Guillain-Barresyndrome (0.1%). It is acquired mainly from eating poultry. Incidence isabout 2.5 million persons/year in USA. C. jejuni accounts for 99% ofcases. It can vary from sub-clinical to severe in compromised patients.It is usually untreated with only fluid replacement or, if the diseaseis severe or threatening, with antibiotics (Erythromycin, tetracyclineor fluoroquinolone).

TABLE 19 MIC/MFC (μg/mL) for Compound (1) (24 h, 35° C., O₂ - micromethod) Bacteria MIC MMC Neisseria gonorrhoeae 2 2 Haemophilusinfluenzae 0.125 0.125 Streptococcus pneumoniae 2 2

The above are significant human pathogens, all of which have successfultreatment regimens with antibiotics. N. gonorrhoeae is a cause ofvaginitis in women. Compound (1) is thus active at low concentrationsagainst two causes, Candida albicans and N. gonorrhoeae.

Trichomonas vaginalis

Method

The MIC of clinical isolate of Trichomonas vaginalis was determined bymacrobroth dilution in Diamond's complete medium, modified by Klass(Modified TYM) as described by Garcia, L. Cultures were contained in 5mL glass, screw-capped bottles without air-spaces. Volumes of 5 mL oflog 2 dilutions, from 512 μg/mL Compound (1) in 5% DMSO to 0.25 μg/mLCompound (1) in 0.002% DMSO in modified TYM, were inoculated with 0.5 mLvolume of cells in log phase of growth giving a final inoculum densityof 1×10⁴ to 3×10⁴ cell/mL. Bottles were incubated aerobically at 37° C.for 24 h before microscopic examination of motility. MIC was determinedas the lowest concentration showing no motility. Aliquots of 0.5 mL fromall tubes showing no motility were subcultured into further 5 mL volumesof modified TYM and incubated aerobically at 37° C. for up to 5 days toconfirm non-viability.

Tests were validated by growth controls in TYM, modified TYM with 2.5%DMSO and modified TYM with 5% DMSO.

Tests were performed as 3 replicates on different days.

Results

MIC MMC Trichomonas vaginalis 4 μg/mL 4 μg/mL

Example 24 Development of Resistance to Compound (1)

Selected resistant strains from the 12 week resistance testing wereretested simultaneously with parent strains using the new microdilutionmethod.

TABLE 20 Change in MIC (μg/mL) of fungal strains after 12 weekscontinuous exposure to sub-inhibitory concentrations of Compound (1) inMHB MIC μg/mL M38-P Test strains Initial Final Rhodotorula rubra 8 16Aspergillus fumigatus 8 8 Rhizopus stolonifer 4 16 Fusarium graminearum4 4

Fungi exhibit up to a four-fold difference in MIC on repeat testing.Significant increases in MIC are ≧8-fold.

There is no significant development of resistance by the filamentousmould strains tested.

Example 25 Effect of Formulation in Ethanol on Activity

The effect of formulation in DMSO and ethanol on the MIC of Compound (1)was compared for Candida albicans, Salmonella Typhimurium andEscherichia coli using both the macrodilution and microdilution tests.

Stock solutions in ethanol were allowed to stand for 48 h before use toimprove solubility. Compound (1) is not as soluble in ethanol as inDMSO. The concentration of ethanol and DMSO was kept constant at 2% andcompared to a decreasing concentration of solvent on normal dilution ofthe stock solution. There was no difference between the two testmethods. There was no difference in MIC between constant and decreasinglevels of DMSO. Only E. coli showed an enhanced susceptibility toethanol in the presence of a constant 2% ethanol.

TABLE 21 Effect of solvent on MIC (μg/mL) of Compound (1) usingmicrodilution DMSO EtOH (2%) MIC MBC MIC MBC Escherichia coli 512 >512128 256 Salmonella 512 >512 512 >512 Typhimurium Candida albicans 8 16 816

The synergistic effect of ethanol on E. coli was noted previously whensolvents were being tested for selection of an appropriate solvent forthe drug. Ethanol has no enhancing effect on Staphylococcus, Salmonellaor Candida. DMSO is a better solvent for the drug in in-vitro tests.Ethanol will be used for animal studies.

Example 26 Stability of Compound (1) Solutions on Storage

Stock solutions of Compound (1) at 512 μg/mL in water+5% DMSO werestored at room temperature (RT˜18-21° C.), 4° C. and −20° C. for up to12 weeks and the potency tested by measurement of MIC for Bacillussubtilis at 2-weekly intervals.

TABLE 22 Stability of Compound (1) stock solutions at 512 μg/mL testedby measurement of the MIC/MBC for Bacillus subtilis (24 h, 35° C.) at 2weekly intervals RT₁ 4° C. −20° C.₂ Test time MIC MBC MIC MBC MIC MBC 04 8 4 8 4 8 2 4 8 4 8 4 8 4 4 8 4 16 4 16 6 4 16 4 16 4 16 8 8 16 4 16 416 10 8 16 4 16 4 16 12 8 16 4 16 4 16 ₁Solution changed from lightyellow to dark yellow after 8 weeks. ₂Solution changed from light yellowto dark yellow after 2 weeks.

Compound (1) is very stable, dilute solutions retaining potency for 12weeks on storage at 4° C. and −20° C. Twofold loss of potency at roomtemperature after 6 weeks is very low compared to working solutions ofantibiotics. It is also within the allowed variation range for MICmeasurements for bacteria (2-fold). Control antifungals were not tested.

Example 27 Effect of Compound (1) on Growth of the Human MalariaParasite, Plasmodium Falciparum, in Human Red Blood Cells In Vitro

The aim of this example was to quantify the effect of Compound (1) oninvasion and growth of the human malaria parasite Plasmodium falciparumin human red blood cells in vitro.

Methods

Malaria Parasites

3D7 is a well characterised in vitro culture-adapted line of P.falciparum that was used for these experiments. The parasite undergoesrepeating cycles of growth and replication within human red blood cells.The duration of each complete cycle is 48 hours, beginning with youngring-stage parasites which mature through pigmented trophozoites duringthe first 24 hours of the cycle to segmented schizonts which burst torelease infectious merozoites which rapidly invade red blood cells.Newly invaded merozoites become ring forms, and the cycle continues.

Parasite Culture and Growth Inhibition Assays

P. falciparum parasites were maintained in synchronous in vitro culturein freshly collected human red blood cells, using well-establishedtechniques. For invasion assays, red blood cells containingstage-synchronized mature, pigmented trophozoites were purified andresuspended in fresh human red blood cells, so that approximately two inevery 100 red blood cells was parasitised (2% parasitaemia). Freshculture media was added to give a final red blood cell concentration of2×10⁸ red cells/ml.

Aliquots of the red blood cell suspension containing either the testcompound, the vehicle alone (in this case EtOH) or PBS (control) wereincubated at 37° C. in an atmosphere of reduced oxygen tension (1% O₂,3% CO₂, 96% N₂). Thin blood smears were made immediately (time=0) thensubsequently after 24, 48 and 72 hours of culture. For each smear,parasitaemia and stage of parasite maturation was quantified bymicroscopic examination after staining with Giemsa at pH 7.2. Thisallowed invasion, parasite development and subsequent re-invasion to bequantified. At each sampling time point, the culture medium(±compound/vehicle) in all samples was completely replaced with freshmedium.

Compound (1) was tested as aqueous solutions of 100, 400 and 1000 μg/mleach containing 10% EtOH. Stock solutions were stored at 4° C. untilrequired. For the assay, each solution was further diluted 1:40 incomplete parasite culture medium (pH 7.2) to the desired workingconcentration (5, 10 and 25 μg/ml), then sterile filtered (0.22 μm)before being added to the parasitised red blood cell suspension. Stocksolutions were stored at 4° C. throughout the duration of the assay, anddiluted appropriately in parasite culture medium when required. Itshould be noted that at 1000 μg/ml, the compound was incompletelysoluble, even after warming to 37° C. and vigorous vortexing. Thus thetests performed at a putative concentration of 25 μg/ml, may in facthave been performed at a lower effective concentration.

Results

The effect of Compound (1) on parasite growth was tested at finalconcentrations of 5, 10 and 25 μg/ml. Results are presented graphicallyin FIG. 2. A concentration-dependent inhibitory effect on parasitegrowth and replication was detected at all concentrations of drugtested, being greatest at the highest concentration tested (25 μg/ml)after 72 hours of culture. EtOH alone, at a final concentration of 0.25%had no significant effect on parasite growth. All concentrations of thecompound tested showed no detectable adverse effect on red blood cellmorphology.

At 25 μg/mL no parasites were observed and at 10 μg/mL only very fewwere found, suggesting that the compound actually killed the parasites.

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and areincorporated herein by this reference.

REFERENCES

-   Burton, H., Duffield, G., J. Chem. Soc., 1949, 78-   Denisenko P. P., Tarasenko A. A., Russian patent No. 2145215,    “Substances having antimicrobial, antifungal, antiprotozoal    activity” published 10 Feb. 2000-   Foyer, G., Chemistry of nitro and nitroso groups, Moscow, 1973, Pt.    2, pp. 194-195-   Garcia, L., Parasite culture: Trichomonas vaginalis, Clinical    Microbiology Procedures Handbook, H. D. Isenberg (ed.), volume 2,    American Society for Microbiology, Washington, USA, 7.9.3.1-7.9.3.6.-   Hamlin, K., Weston, A., J. Am. Chem. Soc. 71, 2210 (1949)-   Knoevenagel, E., Walter, L., Ber., 37, 4502 (1904)-   Kuna P., Chemical radiation protection, Moscow, 1989, pp. 25-28-   Mashkovskiy M. D., Clinical agents, Pt. 2, Moscow, 1986, p. 189-   Perekalkin V. V., Unlimited nitrocompounds, Leningrad, 1982, pp. 55,    59, 61, 71, 73, 88, 89, 91, 95-   Perekalkin V. V., Unlimited nitrocompounds, Leningrad, 1982, p. 67-   Perekalkin V. V., Unlimited nitrocompounds, Moscow, 1966, p. 119-   Vladimirov V. G. et al., Radiation protectors, structure and    operation, Kiev, 1989, p. 139

1. A method for the therapeutic treatment of a Plasmodium falciparum orTrichomonas vaginalis infection, the method comprising administering toan animal in need thereof an effective amount of a compound of generalformula I:

in which X and Y are either the same or different, and are each aheteroatom selected from the group consisting of O, N, and S;

 is a double or single bond depending on the heteroatoms X and Y; R₁ toR₅ are either the same or different and selected from hydrogen or anon-deleterious substituent; and R₆ and R₇ are either the same ordifferent, and selected from hydrogen and a non-deleterious substituent,or one of R₆ and R₇ are absent when there is a double bond present, or apharmaceutically-acceptable salt thereof.
 2. The method of claim 1, inwhich X and Y are O, R₁ is methyl, and R₂ and R₃ are hydrogen.
 3. Themethod of claim 1, wherein the compound is3,4-methylenedioxy-β-methyl-β-nitrostyrene,3,4-methylenedioxy-β-nitrostyrene, or a combination thereof.
 4. Themethod of claim 1, wherein the infection comprises a Trichomonasvaginalis infection.
 5. The method of claim 1, wherein the infectioncomprises a Plasmodium falciparum infection.
 6. The method of claim 1,wherein the animal is a mammal.
 7. The method of claim 6, wherein themammal is human.
 8. The method of claim 1, wherein the compound iscomprised within a pharmaceutically-acceptable composition that furthercomprises one or more carriers, adjuvants or vehicles.
 9. The method ofclaim 8, wherein the composition is administered to the animal orally,topically, or parenterally.
 10. The method of claim 9, wherein thecomposition is administered to the animal parenterally by subcutaneous,intravenous, intramuscular, intrathecal, or intracranial injection orinfusion.
 11. The method of claim 8, wherein the composition isformulated for veterinary administration.
 12. The method of claim 1,wherein treatment of the infection comprises relieving or amelioratingone or more effects of the infection, or arresting the development ofone or more diseases caused by the infection.
 13. A method for relievingor ameliorating one or more effects of a Plasmodium falciparum orTrichomonas vaginalis infection in an animal, the method comprisingadministering to an animal in need thereof a composition comprising aneffective amount of a compound of general formula I:

in which X and Y are either the same or different, and are each aheteroatom selected from the group consisting of O, N, and S;

 is a double or single bond depending on the heteroatoms X and Y; R₁ toR₅ are either the same or different and selected from hydrogen or anon-deleterious substituent; and R₆ and R₇ are either the same ordifferent, and selected from hydrogen and a non-deleterious substituent,or one of R₆ and R₇ are absent when there is a double bond present, or apharmaceutically-acceptable salt thereof.
 14. The method of claim 13,wherein the animal is human.
 15. A method for arresting the developmentof a Plasmodium falciparum or Trichomonas vaginalis infection in ananimal, the method comprising administering to an animal in need thereofa composition comprising a therapeutically-effective amount of acompound of general formula I:

in which X and Y are either the same or different, and are each aheteroatom selected from the group consisting of O, N, and S;

 is a double or single bond depending on the heteroatoms X and Y; R₁ toR₅ are either the same or different and selected from hydrogen or anon-deleterious substituent; and R₆ and R₇ are either the same ordifferent, and selected from hydrogen and a non-deleterious substituent,or one of R₆ and R₇ are absent when there is a double bond present, or apharmaceutically-acceptable salt thereof.
 16. The method of claim 15,wherein the animal is human.